The present invention relates generally to optical imaging, and more particularly to background noise reduction in fluorescence imaging systems.
Fluorescence imaging typically involves illuminating a fluorescent target with light having wavelength content that matches, at least partially, the absorption spectrum of the fluorescence label and imaging the target with an optical detection system that favors the emitted fluorescence light over any reflected or scattered portion of the excitation light. Like any other detection system, the performance of a fluorescence imaging system can be described by a Signal-to-Noise Ratio (SNR) for a given fluorescence concentration at the target plane. The goal of an optical design is to maximize the fluorescence signal for a given concentration of fluorescence emitting material detected by the imaging system and at the same time minimize its noise level. Elements that contribute to a high SNR for a CCD-based fluorescence imaging system are reflected in the following equation
      S    ⁢                  ⁢    N    ⁢                  ⁢    R    =            S      N        =                            S          Fl                ⁡                  (                      P            ,            t                    )                                                                N              Dark              2                        ⁡                          (              t              )                                +                                    N              Exc              2                        ⁡                          (                              P                ,                t                            )                                +                                    N              AutoFluor              2                        ⁡                          (                              P                ,                t                            )                                          where, SF1(P,t) is the fluorescence signal from a desired target and varies at low concentrations linearly with the power of the excitation light, P, and CCD exposure time, t, NDark2 (t) is the dark background signal, i.e. when the excitation light is turned off. NExc2(P,t) is the detected optical background signal resulting from excitation light reflected and/or scattered and leaking through the emission filtering system and NAutoFluor2(P,t) is the detected optical background signal resulting from amounts of excitation light being absorbed by fluorescence-mounting media that itself fluoresces in the passing band of the detection filtering system.
High SNR requires maximizing the detected signal and at the same time minimizing each of the background components. Given an imaging system with efficient light collection and CCD conversion efficiencies, the detected fluorescence signal can be maximized by increasing the light excitation power and/or increasing the exposure time. Equally important, though, is the elimination and/or reduction of the detected background levels that contribute to noise. For CCD-based imaging, NDark2 (t) consists primarily of two components: read-noise, which does not change as P and t change, and dark-current which does depend on exposure time, t. The former is typically kept low by proper selection of the CCD sensor, the speed of reading out charges from that sensor, and the electronics design around the sensor. The dark-current component is primarily a property of the CCD chip itself and is typically kept under control by properly cooling the CCD. Therefore, NDark2 (t) is primarily set by the design of the camera part of the detection system and typically sets the minimum level of noise in the system, even if there is no excitation light or fluorescence signals. And, a sensitive system needs to have low NDark2 (t) to begin with. If, then, the other two components that contribute to noise are completely eliminated, the SNR can be indefinitely increased by increasing P and t. In reality, there will be other limitations, such as photo-bleaching, safety, availability of sources, etc., that limit the increase in P. Even with such limitations, a better design is a design that has no or minimum levels of NExc2(P,t) and NAutoFluor2(P,t).
U.S. Pat. No. 7,286,232 establishes an innovative method for reducing NExc2(P,t) significantly in CCD-based wide area imaging. The elements of the patented filtering system were designed to collectively suppress NExc2(P,t) to levels much lower than prior art and do so across the whole, relatively large, field of view. It was shown that for the application of imaging mice, which are known to have significant levels of auto-fluorescence, NExc2(P,t) was suppressed by the invention well below NAutoFluor2(P,t) and the resulting noise is then limited by the auto-fluorescence of the target itself, i.e. the mouse.
Imaging in the Near-InfraRed (NIR) wavelength range has recently become the focus of a lot scientific work because of low auto-fluorescence of tissue and other sample-holding media. By reducing the auto-fluorescence of the target itself, demand becomes more stringent on the imaging system itself to not produce optical background levels that can be the limiting factor.
Therefore it is desirable to provide systems and methods that overcome the above and other problems and that allow for maximizing the performance of fluorescence imaging and thus the information that Scientists can use.